Long-lived cells latently infected with HIV-1 form a viral reservoir that cannot be eliminated by current anti-retroviral therapy regimens, even when those therapies are applied over an infected person's lifespan. A comprehensive understanding of cellular and tissue-based reservoirs would facilitate the development of new approaches to eradicate this persistent viral reservoir. We hypothesize that brain and immune tissue macrophage-lineage cells constitute a significant HIV-1 reservoir that is genetically and phenotypically distinct from the CD4+ T cell reservoir. Highly sensitive methods will be used to quantify HIV-1 nucleic acids present in macrophage lineage cells in brain, CSF, and immune tissue and to also determine whether astroglia contribute to a non-T cell reservoir. A key issue is whether macrophage-lineage cells are actually infected, or whether detectable HIV-1 nucleic acids represent ingested or contaminating T cells. Using highly purified macrophages, we will ascertain whether they carry full length, intact proviruses, actively express HIV-1 mRNA, and can produce infectious virus. We will also establish whether macrophage proviruses can be activated ex vivo by latency reversing agents (LRAs). Finally, we will use PacBio next generation sequencing (NGS) and single genome analyses (SGA) to investigate phylogenetic relationships and compartmentalization of HIV-1 in brain, CSF, and immune tissues and in specific cell types i.e. purified macrophages, astroglia and T cells. We propose the following three aims: Aim 1. To measure and characterize the HIV-1 reservoir in macrophages from brain and immune tissue. HIV-1 proviral DNA and mRNA will be quantified in macrophage-lineage cells and T cells purified from brain, CSF, and immune tissue to better understand the number and distribution of cells that are infected and expressing HIV-1 mRNA. Aim 2. To evaluate the presence of replication competent HIV-1 in macrophages from brain and immune tissue. PCR and culture protocols will evaluate whether intact, integrated, replication-competent proviruses are present in macrophages and can be activated with LRAs. Aim 3. To investigate the phylogenetic relationships of HIV-1 in macrophages and T cells in brain and immune tissue. Single genome PCR and PacBio deep sequencing of env and pol genes will be used to investigate the relationships between HIV-1 found in macrophages and T cells in brain, CSF, and immune tissue. Our proposal aims to (1) determine whether macrophages in immune tissue are infected with HIV-1 and form a viral reservoir, (2) set up approaches to quantify infectious HIV-1 proviruses in monocyte/ macrophages in brain, CSF and immune tissue and (3) establish phylogenetic relationships between HIV-1 populations present in purified macrophages and T-cells present in different tissues. Study results will be important for informing strategies to achieve remission, and potential cure, in HIV-1 infected individuals.